Bacillus Sp. Biosurfactants, Composition Including Same, Method for Obtaining Same, and Use Thereof

ABSTRACT

Biosurfactants produced by a strain of  Bacillus  sp and to uses thereof. A composition comprising the biosurfactants, and a method for producing the biosurfactants. A method for obtaining a biosurfactant, and a device for implementing the method. The production of biopesticides or biosurfactants for the phytosanitary industry, and in the fields of the food, cosmetics pharmaceutical and oil industries and the environment.

BACKGROUND Technical Field

The present invention relates to a strain of Bacillus sp.

The present invention also relates to biosurfactants produced by astrain of Bacillus sp and to uses thereof. It also relates to acomposition comprising these biosurfactants, as well as a method forproducing these biosurfactants.

The present invention also relates to a method for obtaining abiosurfactant, as well as to a device for implementing this method.

The present invention finds in particular applications in the productionof biopesticides or biosurfactants for the plant health industry, andalso in the fields of the food, cosmetics, chemical, pharmaceutical andoil industries and the environment.

In the following description, the references between ([ ]) refer to thelist of references presented at the end of the examples.

Prior Art

The conventional agricultural production system uses plant-healthproducts of the pesticide type in order to ensure sufficient productionin terms of quantity and quality, in accordance with the expectations ofthe markets and at a cost acceptable to the consumer. Though the use ofthese products affords benefits for the agricultural systems, it maynevertheless give rise to negative effects for human health and for theenvironment. Degradation of the quality of subterranean water andsurface water and reduction in biodiversity in the agriculturalenvironment are the consequences most frequently cited.

Biosurfactants, in particular of bacterial origin, are known to havenumerous interesting properties, in particular surfactant, antiviral,antibacterial and antifungal properties able to be exploited in theplant-health field. These biosurfactants can be used alone or in amixture of several biosurfactants. Synergetic effects have been shownwhen the biosurfactants are used in the form of mixtures (Ongena andJacques, 2008 Bacillus lipopeptides: versatile weapons for plant diseasebiocontrol. Trends Microbiol. 16, 115-125 [1]; CZ20011620 [2]; DE102005050123 [3]).

These biosurfactants have better biodegradability, lower toxicity andgreater physicochemical resistance compared with a pesticide of chemicalorigin. Moreover, the cosmetic market has a particular stake inmolecules of biological origin, which combine antimicrobial activitiesand physicochemical properties such as emulsifiers.

Biosurfactants are also used in the assisted recovery of oil containedin deposits, where the injection of biosurfactants reduces the viscosityof the oil and substantially improves the proportion of oil recovered.They are also used for combating the pollution of water by hydrocarbonsand are much more effective than chemical surfactants. Furthermore,these biosurfactants are not toxic for the ecosystem of the watertreated.

The demand for biosurfactants has therefore increased over the past fewyears, in particular in the food, cosmetics, chemical, pharmaceuticaland oil industries and the environment. Many production methods havebeen studied and used and have been the subject of publications orpatent application filings (FR 2578552 [4]).

However, the biosurfactants currently available are not very effectiveand have limited biological and/or chemical properties.

There therefore exists a real need to provide alternativebiosurfactants, preferably having improved properties compared with thebiosurfactants of the prior art.

Moreover, there currently exists a real need to have available effectivemeans for obtaining biosurfactants.

The methods for producing biosurfactants produced by Bacillus sp. havebeen particularly studied. However, these methods lead to the formationof foam caused by the addition of oxygen, in the form of bubbles. Afirst approach is to use aerated reactors that are mechanically agitatedand to continuously extract the foam caused by the biosurfactant andcontaining the latter. This method is laborious and not very open touse, in particular on a large scale (Guez et al., 2007. Setting up andmodelling of overflowing fed-batch cultures of Bacillus subtilis for theproduction and continuous removal of lipopeptides. J Biotechnol, 131,67-75 ([5]).

In order to avoid this problem of foam, attempts have been made to workunder anaerobic conditions and to use nitrate as the final electronacceptor (Davis, Lynch and Varley. 1999. The production of surfactin inbatch culture by Bacillus subtilis ATCC 21332 is strongly influenced bythe conditions of nitrogen metabolism. Enzyme Microb. Technol. 25,322-329. [6]; WO 0226961 [7]; EP 1320595 [8]). Productions ofbiosurfactants depend greatly on the ability of the strains to adapt ornot to these anaerobic conditions. No effective solution making itpossible to produce biosurfactants in industrial quantities has beendeveloped up to the present time. Moreover, the use of pesticides ofchemical origin being more and more contested, it is necessary toresearch and use molecules of biological origin in order to replacepesticides of chemical origin and to develop methods for producing thesemolecules of biologic origin on an industrial scale.

There therefore exist real requirements to develop a method and deviceovercoming these defects, drawbacks and obstacles of the prior art,including a method for continuously producing biosurfactants in largequantities with low production costs.

SUMMARY

The present invention precisely meets the aforementioned requirements,by providing a Bacillus subtilis strain, mycosubtilins, a compositioncomprising these mycosubtilins, and a method for obtaining thesemycosubtilins.

The present invention also provides a method and device for producing abiosurfactant on an industrial scale, in particular by eliminating orlimiting the formation of foam.

The subject matter of the present invention is thus a method forobtaining a biosurfactant, comprising a step (a) of culture of amicroorganism capable of producing a biosurfactant in a culture mediumcomprising an organic substrate, the culture of the microorganism beingperformed on the surface of an air/liquid membrane contactor.

The inventors are in fact the very first to have implemented this methodand have discovered, surprisingly, that the immobilisation of the cellson the air/liquid membrane contactor is particularly favourable to theproduction of biosurfactants continuously, while avoiding the formationof foam. Furthermore, the method according to the invention increasesthe biosurfactant production yield. In addition, the use of anair/liquid membrane contactor in the method according to the presentinvention makes it possible to produce a biosurfactant continuously.

Hereinafter, “biosurfactant” means a surface-active molecule that isamphiphilic and is produced from a microorganism. It may for example bea compound chosen from the group comprising a lipopeptide, aphospholipid, a glycolipid, a lipoprotein, or a fatty acid ester. Forexample, the lipopeptide is chosen from the group comprising an iturin,a surfactin, a mycosubtilin, a syringomycin, a fengycin (orplipastatin), a lichenysin, a bacillomycin, a kurstakin, a tolaasin, anarthrofactin, a serrawettin, a putisolvin and a massetolide. Thephospholipid may for example be chosen from the group comprising aphosphatidylcholine. The ester may for example be chosen from the groupcomprising a sorbitan or rhamnose ester, a monomyristin, a monolinoleinand a monolinolenin. The glycolipid may for example be a rhamnolipid.

“Culture of a microorganism” means all the techniques used for growing amicroorganism and/or making it produce one or more molecules.

“Microorganism capable of producing a biosurfactant” means anyunicellular or pluricellular microscopic organism devoid of tissuedifferentiation, and having the ability to synthesise a biosurfactant.It may for example be a bacterium, a yeast, a mould or an alga.

For example, the bacterium may belong to the genus chosen from the groupcomprising Bacillus, Pseudomonas, Rhodococcus, Acinetobacter, Serratia,Burkholderia, Mycobacterium, Nocardia, Flavobacterium, Corynebacterium,Clostridium, Thiobacillus, Arthrobacter, Alcanivorax and Paenibacillus.For example, the yeast may belong to a genus chosen from the groupcomprising Candida, Pseudozyma, Ustilago, Schizonella, Kurtzmanomyces,Torulopsis, Rhodotorula and Wickerhamiella. Preferably, themicroorganism capable of producing a biosurfactant belongs to the genusBacillus.

When the microorganism capable of producing a biosurfactant belongs tothe genus Bacillus, it may for example be chosen from the groupcomprising Bacillus subtilis, Bacillus thuringiensis, Bacilluslicheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacilluspumilus and Bacillus mojavensis. It may for example be a case of strainschosen from the group comprising Bacillus subtilis, such as the onefiled on 10 Mar. 2011 under the number CNCM I-4451 in the NationalCollection of Microorganism Cultures (CNCM) of the Institut Pasteur(Paris, France). Also called Bacillus subtilis BBG125, as well asBacillus subtilis ATCC 21332, Bacillus subtilis BBG21, Bacillus subtilisATCC 6633, Bacillus subtilis BBG100, Bacillus subtilis ATCC 9943,Bacillus subtilis S499, Bacillus subtilis BBG116, Bacillus subtilisBBG131, Bacillus licheniformis BAS50, a strain derived from Bacilluslicheniformis ATCC 14580 and Bacillus thuringiensis BBG300.

Preferably, for producing micosubtilin, the microorganism capable ofproducing a biosurfactant is chosen from the group comprising Bacillussubtilis BBG125, Bacillus subtilis BBG100 and Bacillus subtilis BBG1116,preferably from the group comprising Bacillus subtilis BBG125 andBacillus subtilis BBG100.

Preferably, for producing surfactin, the microorganism capable ofproducing a biosurfactant is Bacillus subtilis BBG131.

Preferably, for producing fengycin, the microorganism capable ofproducing a biosurfactant is chosen from the group comprising Bacillussubtilis ATCC 21332 and Bacillus subtilis BBG21.

Preferably again, according to the invention, the microorganism capableof producing a biosurfactant is Bacillus subtilis BBG125.

When the microorganism capable of producing a biosurfactant belongs tothe genus Pseudomonas, it may for example be chosen from the groupcomprising Pseudomonas aeruginosa, Pseudomonas cichorii, Pseudomonasputida, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonassyringae and Pseudomonas tolaasii.

Hereinafter, “organic substrate” means any substance or mixture ofsubstances making it possible to grow the microorganism and/or to makeit produce one or more molecules. For example, the organic substrate maybe chosen from the group comprising starch, glucose, glutamate,saccharose, xylose, glycerol, the organic acids, amino acids and amixture of these organic substrates. For example, the organic substratemay be the Landy medium with the following composition: glucose, 20g/litre; glutamic acid, 5 g/litre; yeast extract, 1 g/litre, K₂HPO₄, 1g/litre; MgSO₄, 0.5 g/litre, KCl, 0.5 g/litre, CuSO₄, 1.6 mg/litre;Fe₂(SO₄)₃, 1.2 mg/litre, MnSO₄, 0.4 mg/litre (Landy et al. 1948.Bacillomycin; an antibiotic from Bacillus subtilis active againstpathogenic fungi. Proc. Soc. Exp. Biol. Med. 67, 539-541) [9]. Theorganic substrate may also for example be a modified Landy medium, forexample a Landy medium supplemented with ammonium sulfate at 2.3 g/litreand/or the glutamic acid concentration is 2 g/litre (Guez et al, 2008.Respiration activity monitoring system (RAMOS), an efficient tool tostudy the influence of the oxygen transfer rate on the synthesis oflipopeptide by Bacillus subtilis. J. Biotechnol. 134, 121-126 [10]).

Hereinafter, “air/liquid contactor” means a device for oxygenating aliquid medium using a gas containing oxygen. An air/liquid contactorcomprises in particular an air/liquid membrane contactor. It may forexample be a case of a reactor comprising an air/liquid membranecontactor. By way of examples of air/liquid contactor, the followingcontactors can be mentioned: “Hollow fibre cartridges” from GEHealthcare in accordance with the models defined by referencescommencing with CFP, and “Hollow fibre modules” from Spectrum Labs inaccordance with the models defined by the references commencing with KM.

An air/liquid membrane contactor also separates the liquid phase fromthe gaseous phase: the gaseous phase circulates on one side of themembrane and the liquid phase flows on the other side (Remize andCabassud 2003, A novel bubble-free oxidation reactor: the G/L membranecontactor. Recent progress in method engineering. Integration ofmembranes in the methods 2. Lavoisier Tec and Doc. [11]), without thesetwo phases mixing.

“Air/liquid membrane contactor” means a porous membrane allowing thediffusion of oxygen in a culture medium.

For example, the air/liquid membrane contactor may be a membrane madefrom hollow fibres or flat membranes.

The air/liquid membrane contactor may for example have pores with a sizeof between 0.01 and 2 μm, for example between 0.01 and 1 μm, for examplebetween 0.1 and 0.65 μm.

The air/liquid membrane contactor may for example have a surface area ofbetween 0.1 and 20 m². The surface area of the air/liquid membranecontactor is preferably greater than 1 m².

The air/liquid membrane contactor may for example be a hydrophobicmembrane, guaranteeing better separation of these two phases. Forexample, the air/liquid membrane contactor may be produced from amaterial chosen from the group of polymers comprising polyethersulfone,polypropylene, polysulfone, regenerated cellulose and cellulose esters.

The air/liquid membrane contactor may for example be flat, cylindrical,cylindroconical or any geometric shape optimising the microorganismculture and exchanges with oxygen.

By way of example of air/liquid membrane contactors, the followingmembranes can in particular be mentioned:

-   -   (CFP-6-D-45) from GE-Healthcare Europe GmbH (Munich, Germany),    -   hollow-fibre filtration modules (Hollow fibre cartridges) from        GE Healthcare in accordance with the models defined by the        references commencing with CFP,    -   hollow fibre filtration modules (Hollow fibre modules) from        Spectrum Labs in accordance with the models defined by the        references commencing with KM.

According to the invention, the microorganisms capable of producing abiosurfactant may be immobilised actively either completely or partiallyon the surface of the air/liquid membrane contactor. In other words, amajor part of the microorganisms present in the air/liquid contactor areimmobilised on the surface of the membrane of this contactor, the otherpart being in suspension in the culture medium after release thereof.

Thus the method according to the invention may comprise a step (a) ofculturing a microorganism capable of producing a biosurfactant in aculture medium comprising an organic substrate, the microorganism beingcultured on the surface of an air/liquid membrane contactor. In otherwords, the method of the present invention advantageously makes itpossible to dispense with a culture dish or a fermenter. The methodaccording to the invention advantageously makes it possible to producemycosubtilins continuously and in highly satisfactory quantities.Advantageously, the method of the present invention is implementedcontinuously. A culture dish or fermenter may be added optionally but isnot essential. The addition of a culture dish or a fermenter is ratherinadvisable since it would reduce the production yields. Thus,advantageously, the method of the present invention is implemented in adevice not comprising a culture dish or a fermenter. In other words,according to the method of the invention, the culture of themicroorganism capable of producing a biosurfactant can be carried out onthe surface of the air/liquid membrane contactor only.

The oxygen necessary for the microorganisms capable of producing abiosurfactant is transferred by diffusion through the pores of theair/liquid membrane contactor where said microorganisms are immobilised.In other words, according to the invention, the oxygenation of themicroorganism medium is not done by a bubbling system situated in areactor nor by an oxygenation system situated outside the microorganismculture reactor.

The oxygen flow of the air/liquid membrane contactor can be adjusted toany flow rate making it possible to oxygenate the microorganisms capableof producing a biosurfactant. A person skilled in the art is able todetermine the oxygen flow rates of the air/liquid membrane contactoraccording to the required addition of oxygen. The inventors have foundthat an aeration flow of between 0.2 and 2 volumes of air per volume ofliquid per minute (vvm) is particularly effective for oxygenating theculture medium and the microorganisms. The air flow of the air/liquidcontactor can therefore for example be between 1.5 and 2 vvm.Preferably, the aeration flow of the air/liquid contactor is 0.25 vvmfor producing mycosubtilin. Preferably, the aeration flow rate of theair/liquid contactor is 1 vvm for producing surfactin. Preferably, theaeration flow rate of the air/liquid contactor is 0.5 vvm for producingfengycin.

According to the method of the invention, the culture step (a) can beperformed on the surface of a plurality of air/liquid membranecontactors. For example, the culture step (a) can be performed on thesurface of two air/liquid membrane contactors, for example 3, 4, 5, 6,7, 8, 9, 10 air/liquid membranes, or even more. A person skilled in theart is able to determine the number of air/liquid membrane contactorsaccording to the quantity of biosurfactant to be produced.

One of the objectives of the present invention is to increase thequantity of microorganisms immobilised on the surface of the air/liquidmembrane contactor. This can be done by increasing the surface area ofthe air/liquid membrane contactor and/or the number of air/liquidmembrane contactors.

When the culture step (a) is performed on the surface of a plurality ofair/liquid membrane contactors, the air/liquid membrane contactors mayfor example be disposed in series or in parallel. Preferably, when theculture step (a) is performed on the surface of a plurality ofair/liquid membrane contactors, the air/liquid membrane contactors aredisposed in parallel.

The method of the present invention may further comprise a step ofseparating the biosurfactant from the culture medium containing it. Thisseparation step may be performed by any means known to persons skilledin the art making it possible to separate a substance contained in aliquid medium thereof.

For example, the step of separating the biosurfactant from the culturemedium containing it may comprise one or more steps, chosen from thegroup comprising microfiltration, ultrafiltration, nanofiltration andcentrifugation.

For example, the step of separating the biosurfactant from the culturemedium containing it comprises the following steps:

-   -   (b) microfiltration of the culture medium obtained at step (a),        for separating the microorganism from the culture medium, and/or    -   (c) ultrafiltration of the culture medium obtained at step (a)        or (b), for separating the biosurfactant from the culture medium        obtained at step (a) or (b).

Preferably, the separation step comprises each of steps (b) and (c).

The steps of microfiltration (b) and ultrafiltration (c) make itpossible to continuously extract the biosurfactant from the culturemedium obtained at step (a) and/or (b).

Combining the air/liquid membrane contactor with the microfiltration (b)and ultrafiltration (c) steps thus makes it possible to continuouslyproduce and extract a biosurfactant from a microorganism capable ofproducing it.

The microfiltration step (b) can be performed with any microfiltrationmeans making it possible to separate the microorganism from the culturemedium containing it. For example, the microfiltration means may be amicrofiltration membrane. For example, the microfiltration means may bea microfiltration membrane. For example, the microfiltration means maybe an organic or mineral microfiltration membrane, for example ahollow-fibre membrane.

The microfiltration step (b) may for example be performed with amembrane made from hollow fibres having pore sizes from 0.1 to 0.45micrometres (μm). Preferably, the hollow-fibre membrane used at step (b)has a pore size of 0.2 μm.

By way of example of membranes that can be used for performing themicrofiltration step (b), the following membranes can be cited:

-   -   hollow polysulfone or polyethersulfone fibres with a pore size        of 0.2 μm, reference CFP-2-E-4X2MA (GE-Healthcare Europe GmbH,        Munich, Germany),    -   hollow polysulfone or polyethersulfone fibres with a pore size        of 0.45 μm, reference CFP-4-E-4X2MA or a pore size of 0.56 μm        reference CFP-2-E-6X2MA (GE-Healthcare Europe GmbH, Munich,        Germany),    -   a hollow-fibre microfiltration or ultrafiltration module (hollow        fibre cartridges) from GE-Healthcare according to the models        defined by references commencing with CFP,    -   a hollow-fibre filtration module (Hollow fibre modules) from        Spectrum Labs (Rancho Dominguez, Calif., USA) in accordance with        the models defined by references commencing with KM,    -   Sartocon microfiltration cassettes from Sartorius Stedim        (Aubagne, France) in accordance with the models defined by        references commencing with SPC20.

The ultrafiltration step (c) can be performed with any filtration meansmaking it possible to separate the biosurfactant from the culture mediumcontaining it, and to concentrate the biosurfactant. For example, theultrafiltration means may be an ultrafiltration membrane, for example anultrafiltration membrane made from regenerated cellulose.

The ultrafiltration step (c) may for example be performed with amembrane having a cutoff threshold of between 5 and 50 kilodaltons(kDa), for example between 5 kDa and 30 kDa, for example between 5 and20 kDa. The membrane used at step (c) preferably has a cutoff thresholdof 10 kDa.

By way of example of membranes that can be used for performing theultrafiltration step (c), the following membranes can be cited:

-   -   ultrafiltration membrane with cutoff threshold of 10 kDa made        from regenerated cellulose, reference 3051443901E-SW (Sartorius,        Göttingen, Germany),    -   ultrafiltration membrane with a cutoff threshold of 10 kDa made        from regenerated cellulose, reference P2C010001 (Millipore        Headquarters, 290 Concord Road, Billerica, Mass., USA).

The membranes used at step (a), (b) and (c) are preferably sterilisableat 121° C. for 20 minutes.

The method according to the present invention is differentiated from theknown methods for the biological degradation of organic materials bymicroorganisms that excrete biosurfactants in that it is possible torecycle or not all the microorganisms after having removed from them theresidues of organic matter from the culture medium and biosurfactantsproduced. This makes it possible to obtain a high degree ofconcentration of the microorganisms in the bioreactor.

It is also differentiated from the known methods for the biologicaldegradation of organic materials by microorganisms that excretebiosurfactants in that approximately 95% of the biosurfactants producedremain in the culture medium without the least formation of foam.Approximately 5% of the biosurfactants are adsorbed on the air/liquidinterface of the membrane contactor, but may be desorbed for example bywashing the membrane.

The air/liquid membrane contactor can be washed by any means known topersons skilled in the art in order to recover the biosurfactantsproduced and adsorbed at the air/liquid interface of the membranecontactor. For example, the washing of the air/liquid membrane contactormay be performed with one or more washing solutions chosen from thegroup comprising distilled water, an NaOH solution, an NaOCl solution, asodium or potassium hydrogen carbonate solution, or a sodium orpotassium carbonate solution. The washing solution may be brought to anypH making it possible to increase the quantity of biosurfactantsrecovered. For example, the washing solution is brought to a pH=10.

The washing solution may be brought to any temperature making itpossible to increase the quantity of biosurfactants recovered. Forexample, the washing solution is brought to a temperature of between 20°and 50° C.

According to the present invention, the temperature of the culturemedium may be adjusted by any heating means known to persons skilled inthe art. For example, the heating means may be a heat exchanger. Forexample, the heat exchanger may be chosen from the group comprising aU-tube heat exchanger, a heat exchanger with a horizontal tubularcluster, a heat exchanger with a vertical tubular cluster, a spiral heatexchanger, a plate heat exchanger, a Bouhy column, or a block heatexchanger. The heating means is preferably a tubular heat exchanger.

Thus the method according to the invention can be implemented at anytemperature making it possible to produce a biosurfactant from amicroorganism capable of producing it. For example, the method may beimplemented at a temperature between 0° C. and 70° C., advantageouslybetween 20° C. and 37° C. Preferably, the method may be implemented at atemperature of 22° C. for producing mycosubtilin. The method maypreferably be implemented at a temperature of 30° C. for producingfengycin. Preferably, the method may be implemented at a temperature of37° C. for producing surfactin.

Moreover, the method according to the present invention may beimplemented at any pH making it possible to produce a biosurfactant froma microorganism capable of producing it. The pH may be regulated bymeans of the controlled addition of basic solution or acid solution tothe culture medium.

The basic solution may for example be chosen from the group comprisingsoda, potash and ammonia.

The acid solution may for example be chosen from the group comprisingphosphoric acid, sulphuric acid and nitric acid.

The pH may for example be regulated to any value enabling microorganismscapable of producing a biosurfactant to survive. It may for example beregulated to a value between pH 6 and pH 8, preferably to a value of pH7. A person skilled in the art is able to determine the quantities ofbasic solution and acid solution for regulating the pH to a requiredvalue.

The method according to the present invention may advantageously beimplemented continuously, that is to say the supply of the air/liquidmembrane contactor and the extraction of the biosurfactants produced bythe microorganisms can be carried out without interruption. The methodof the present invention may be performed at any hourly rate making itpossible to extract a biosurfactant from a microorganism capable ofproducing it. The rate at which the method can be implemented, that isto say the flow rate of culture medium added to the air/liquid membranecontactor, can easily be adapted by a person skilled in the art. Theinventors have found that conducting the continuous method at a dilutionrate of between 0.05 h⁻¹ and 0.5 h⁻¹ is particularly effective forproducing biosurfactants from microorganisms. The dilution rate isdefined as the supply or extraction rate divided by the culture volume.The method according to the invention can therefore, for example, beperformed at a circulating hourly rate corresponding to a degree ofdilution of between 0.05 h⁻¹ and 0.5 h⁻¹, advantageously at an hourlyrate of 0.1 h⁻¹.

Another subject matter of the present invention is a device forimplementing the method described herein, said device comprising anair/liquid membrane contactor. For example, the device according to theinvention comprises at least one air/liquid contactor comprising anair/liquid membrane contactor.

The air/liquid membrane contactor and the air/liquid contactor may bethose defined above.

The number of air/liquid membrane contactors and air/liquid contactorsmay be as defined above.

The device according to the invention does not comprise any aerationmeans other than the membrane or the plurality of air/liquid membranecontactors. In other words, the device according to the invention doesnot comprise a bubbling system situated in a reactor. It also does notcomprise an oxygenation system situated outside the microorganismculture reactor. The device according to the present invention mayfurther comprise a microfiltration means and/or an ultrafiltrationmeans. The device according to the invention preferably comprises amicrofiltration means and an ultrafiltration means. The microfiltrationmeans and the ultrafiltration means may for example be those describedabove.

Advantageously, the device according to the invention comprises themeans necessary for continuously implementing the method according tothe invention. In other words, the device advantageously comprises ameans for introducing a culture medium and a means for taking off thebiosurfactant produced by the microorganism. Any introduction means andtake-off means known to persons skilled in the art for obtaining adevice for continuously implementing the method according to theinvention may be used.

The device according to the invention may further comprise anevaporation means. Hereinafter, “evaporation means” means any means forconcentrating a biosurfactant in a medium containing it. It may forexample be an evaporation means chosen from the group comprising avacuum evaporator of the Rotavapor VV000 type (Heidolph Instruments GmbH& Co, Schwabach, Germany) and a climbing-film evaporator. The deviceaccording to the present invention may further comprise a heating meansfor regulating the temperature of the culture medium. The heating meansmay for example be the one described above. The heating system may beconnected to the air/liquid contactor via a system of pipes.

“System of pipes” means any means in which a fluid or gas may circulate.For example, the fluid may be a liquid or a gel. The system of pipesmakes it possible in particular to connect together various elements ofthe device according to the invention. For example, the system of pipesmay be any type of flexible or rigid pipework of the silicone type.(Cole Parmer, Vernon Hills, Ill., USA) or made from 316S stainless steel(Swagelok Company, Solon, Ohio, USA).

The circulation of the fluid or gas in the system of pipes may beregulated by one or more pumps and/or one or more valves.

The device according to the present invention may further comprise atleast one pump.

“Pump” means, within the meaning of the present invention, means forimposing a flow rate on a liquid, for example on a culture medium in thedevice of the present invention. It may for example be a peristalticpump, a lobe pump, or a membrane pump. Mention can be made for exampleof the Masterflex L/S peristaltic pumps compact drive model (Cole ParmerVernon Hills, Ill., USA), Millipore Corporation (Millipore, Bedford,Mass., USA), Sartojet pump (Sartorius, Sartorius Stedim France SAS,Aubagne) and Watson-Marlow 323 (Watson Marlow, Falmouth, Cornwall,United Kingdom). Moreover, the pump may be controlled manually orautomatically.

The device according to the present invention may further comprise atleast one valve.

Hereinafter, “valve” means means for stopping or modifying the flow of aliquid, for example of the culture medium in the device of the presentinvention. It may for example be a regulation valve, a “two-state”valve, or a solenoid valve. Mention can be made for example ofregulation valves made from polyvinylidene fluoride (PVDF),polypropylene (PP), perfluoroalkoxy (PFA) or stainless steel. Moreover,the valve may be controlled manually or automatically.

The inventors describe hereinafter the use of the device according tothe invention for implementing the method of the invention.

Another subject matter of the present invention is a Bacillus subtilisstrain obtained from the strain Bacillus subtilis ATTC 6633 in which theoperon srfA coding for the synthetase surfactin has been interrupted andwhere the promoter of the operon myc coding for the micosubtilinsynthetase has been replaced by a constitutive strong promoter P_(repU).

Preferably, this Bacillus subtilis strain is the Bacillus subtilisstrain filed on 10 Mar. 2011 under the number CNCM I-4451 at theNational Collection of Microorganism Cultures (CNCM) of the InstitutPasteur (Paris, France). This strain is also called Bacillus subtilisBBG125.

Another subject matter of the present invention is a method forproducing mycosubtilins comprising a step of culturing a strain ofBacillus subtilis according to the invention and a step of recoveringthe mycosubtilins obtained.

The Bacillus subtilis BBG125 strain may for example be used in anymethod for producing biosurfactants, in particular in a method forproducing C18 and C17 Gln3 mycosubtilins described above. For example,the method for producing biosurfactants may comprise a step of culturingthe Bacillus subtilis BBG125 strain and a step of recovering thebiosurfactants obtained. For example, the method for producingbiosurfactants may the one of the present invention described above.

The Bacillus subtilis BBG125 strain developed by the inventors isparticularly surprising. This strain makes it possible to producemycosubtilins without producing surfactin. Furthermore, it makes itpossible to produce mycosubtilins that have never been described.

Thus the present invention also relates to the following mycosubtilins:

-   -   C18 mycosubtilin: a mycosubtilin the fatty acid chain of which        comprises 18 carbon atoms, and represented by the following        formula (I):

-   -   C17 mycosubtilin Gln3: a mycosubtilin the fatty acid chain of        which comprises 17 carbon atoms and having a glutamine in the        place of asparagine in position 3 in its peptide cycle and        represented by the following formula (II):

The C18 and C17 Gln3 mycosubtilins described above can be used as anantifungal agent. They moreover have antifungal effects equivalent to oreven greater than the mycosubtilins currently used as an antifungalagent.

Hereinafter, “antifungal agents” means substances having the ability totreat and/or prevent infections by fungi and/or yeasts.

The inventors have also produced a composition comprising a mixture ofmycosubtilins.

Thus another subject matter of the present invention is a compositioncomprising at least one C18 mycosubtilin and/or at least one C17 Gln3mycosubtilin.

For example, this composition may further comprise one or more othermycosubtilins chosen from the group comprising an iso-C16 mycosubtilin,an n-C16 mycosubtilin, an anteiso-C17 mycosubtilin and an iso-C17mycosubtilin.

When a C18 mycosubtilin is present in the composition, it may be presentat a concentration of between 1% and 5% by weight of the composition.

When a C17 mycosubtilin Gln3 is present in the composition, it may bepresent at a concentration of between 1% and 20% by weight of thecomposition.

When an iso-C16 mycosubtilin is present in the composition, it may bepresent at a concentration of between 1% and 60% by weight of thecomposition.

When an n-C16 mycosubtilin is present in the composition, it may bepresent at a concentration of between 1% and 10% by weight of thecomposition.

When an anteiso-C17 mycosubtilin is present in the composition, it maybe present at a concentration of between 20% and 95% by weight of thecomposition.

When an iso-C17 mycosubtilin is present in the composition, it may bepresent at a concentration of between 5% and 30% by weight of thecomposition.

For example, the composition according to the invention may comprise, asa percentage with respect to the weight of the composition: between 1%and 60% of iso-C16 mycosubtilin, between 1% and 20% of C17 mycosubtilinGln3, between 1% and 10% of n-C16 mycosubtilin, between 20% and 95% ofanteiso-C17 mycosubtilin, between 5% and 30% of iso-C17 mycosubtilin andbetween 1% and 5% of C18 mycosubtilin.

This composition preferably comprises, as a percentage with respect tothe weight of the composition: 26% of iso-C16 mycosubtilin, 1% of C17mycosubtilin Gln3, 2% n-C16 mycosubtilin, 44% anteiso-C17 mycosubtilin,23% iso-C17 mycosubtilin and 1% C18 mycosubtilin.

This composition may for example be used as an antifungal composition.In other words, it may be a composition for use as an antifungal agent.

The composition comprising a mixture of mycosubtilins according to thepresent invention has an antifungal capability ranging from a minimuminhibiting concentration of 4 to 32 μm.

The mycosubtilins and the composition comprising a mixture ofmycosubtilins according to the present invention can be mixed in asolution chosen from the group comprising water, ethanol, methanol,dimethylsulfoxyde (DMSO), sodium carbonate, Tris-HCl and a mixture ofthese solutions.

The mixture of these solutions may be a binary or ternary mixture. Whenthe mixture of solutions is binary, the water/ethanol, water/methanol,water/DMSO, water/sodium carbonate, water/Tris-HCl, ethanol/methanol,ethanol/DMSO, ethanol/sodium carbonate, ethanol/Tris-HCl, methanol/DMSO,methanol/sodium carbonate or methanol/Tris-HCl ratio may for example bebetween 4/1 and 1/4, for example between 3/1 and 1/3, for examplebetween 2/1 and 1/2, for example a ratio of 1/1, 2/1, 3/1 or 4/1. Whenthe mixture of solutions is ternary, the water/ethanol/methanol,water/ethanol/DMSO, water/ethanol/sodium carbonate, water/ethanolTris-HCl, water/methanol/DMSO, water/methanol/sodium carbonate,water/methanol/Tris-HCl, ethanol/methanol/DMSO, ethanol/methanol/sodiumcarbonate, ethanol/methanol/Tris-HCl or DMSO/sodium carbonate/Tris-HClratio may for example be 1/1/1, 2/1/1, 1/2/1, 1/1/2, 3/1/1, 1/3/1,1/1/3, 3/2/1, 3/1/2, 2/3/1, 2/1/3, 1/2/3 or 1/3/2.

The mycosubtilins and the composition comprising a mixture ofmycosubtilins according to the present invention may for example be usedas an antifungal agent.

The present invention therefore also relates to a mycosubtilin accordingto the invention or a composition according to the invention for use asan antifungal agent.

The inventors therefore provide a method for the continuous productionof biosurfactants making it possible to avoid the formation of foam andto increase the production yields. They have also provided a device forimplementing this method as well as a strain of Bacillus subtilisremarkable in that it is capable of producing, in this method,mycosubtilins that have never been described and which have antifungaleffects that are equivalent or even superior to the mycosubtilinscurrently used as an antifungal agent. The inventors have also providedan antifungal composition having antifungal effects superior to theeffects of the mycosubtilins currently used.

Other advantages may also appear to a person skilled in the art from areading of the following examples, illustrated by the accompanyingfigures given by way of illustration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a device for the continuous production,without the formation of foam, and the extraction of the lipopeptidesproduced by a microorganism. In this figure, M1 represents an air/liquidmembrane contactor made from hollow fibres having the compartments C1and C2 representing respectively an external compartment, in which aircirculates, and an internal compartment in which a culture mediumcirculates. M2 represents the microfiltration member of amicrofiltration means having the compartments C3 and C4. M3 representsthe ultrafiltration membrane of an ultrafiltration means having thecompartments C5 and C6. B1, B2 and B3 each represent scales. Brepresents a motor for driving a stirring means in the dish 2. P1, P2,P3, P4, P5, P6 and P7 represent volumetric pumps. “Mi” means “culturemedium”. “De” signifies “waste” and “Et” signifies “heat exchanger”.

FIG. 2 is a diagram representing a device for the continuous production,without the formation of foam, the extraction and the purification ofthe lipopeptides produced by a microorganism. In this figure, C1, C2,C3, C4, C5, C6, M1, M2, M3, B1, B2, B3, P1, P2, P3, P4, P5, P6, P7,“Mi”, “De” and “Et” have the same meaning as for FIG. 1. M4 representsthe ultrafiltration membrane of an ultrafiltration means having thecompartments C7 and C8. B4, B5, B6 and B7 each represent scales. Brepresents a motor for driving a stirring means in the dish 2 or thedish 4. P8, P9, P10, P11 and P12 represent volumetric pumps. V1, V2, V3,V4 and V5 represent valves. “Co” signifies “condenser” and “X”corresponding to a liquid solution comprising the lipopeptides producedby the microorganism.

FIG. 3 is a diagram representing the device of FIG. 1 in which analternative circuit is shown in broken lines. P13 and P14 representvolumetric pumps. V6, V7 and V8 represent valves. B7 represents scales.

FIG. 4 is a diagram representing the device of FIG. 2 in which analternative circuit is shown in broken lines. P13 and P14 representvolumetric pumps. V6, V7 and V8 represent valves. B7 represents scales.

FIG. 5 shows the first parts of the device described in FIGS. 1 to 4, inwhich there is a plurality of air/liquid contactors that are disposed inparallel. In this diagram, three air/liquid contactors are shown.

FIG. 6 is a schematic representation of the homologous recombination ofa fragment of 2.6 kb, bearing the sequence εpbp-P_(repU)-neo-εfenF ofthe pBGB106 plasmid, the fragments εpbp and εfenF issue by PCR fromBacillus subtilis ATCC 6633, thus forming the plasmid pBG200. In thisfigure, “Sphl, “Xbal”, “BspEl” and “Xmal” represent the restrictionsites of the respective eponymous enzymes. “εpbp” and “εfenF” representcassettes for homologous recombination. “pbp” represents the gene codingfor a protein bonding to penicillin. “P_(myc)” represents the originalpromoter of B. subtilis ATCC 6633. “fenF”, “mycA”, “mucB” and “mycC”represent the four genes that constitute the operon of mycosubtilin.“yngL” represents the gene coding for a protein having an unknownfunction. “P_(repU)” represents the promoter of the replication gene ofpUB110. “neo” represents a gene conferring resistance toneomycin/kanamycin.

FIG. 7 is a schematic representation of the homologous recombination ofa pBG144 plasmid at the srfA operon of the BBG116 strain, leading theBBG125 strain. “EcoRi”, “BstEII” and “Mfel” represent the restrictionsites of the respective eponymous enzymes. “EsrfAA” and “EsrfAA”′represent cassettes for homologous recombination. “hxlB” represents agene situated upstream of the srfA operon. “P_(srfA)” represents thenative promoter of the srfA operon. “cat” represents the gene forresistance to chloramphenicol. “tet” represents a gene for resistance totetracyclin.

DETAILED DESCRIPTION Example 1: Construction of the Bacillus subtilisBBG125 Strain

The Bacillus subtilis BBG125 strain was filed on 10 Mar. 2011 under thenumber CNCM I-4451 in the National Collection of Microorganism Cultures(CNCM) of the Institut Pasteur (Paris, France).

It was constructed from the Bacillus subtilis strain of the ATCC 6633wild type (Duitman et al, 1999. The mycosubtilin synthetase of Bacillussubtilis ATCC 6633: a multifunctional hybrid between a peptidesynthetase, an amino transferase, and a fatty acid synthase. Proc. Natl.Acad. Sci. USA, 96, 13294-13299 [12]) according to the protocoldescribed below.

1.1 Protocol for Constructing the pBG200 Hybrid Plasmid Containingεpbp-P_(repU)-neo-εfenF and rep(R6K)

The pBG106 plasmid (Leclère et al, 2005. Mycosubtilin overproduction byBacillus subtilis BBG100 enhances the organism's antagonistic andbiocontrol activities. Appl. Environ. Microbiol., 71, 4577-4584 [13]),was digested by the restriction enzymes Sphl (Fermentas, Villebon surYvette, France; reference ER0601) and SacI (Fermentas, Villebon surYvette, France; reference ER1131) in order to isolate and purify afragment εpbp-P_(repU)-neo-εfenF of 2.6 kilo-pairs of bases (kb) ofsequence SEQ ID NO: 11, in accordance with the protocol described in:Sambrook and Russell, 2001. Molecular cloning: a laboratory manual,3^(rd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.[14].

This sequence carries two cassettes (εpbp and εfenF) for performinghomologous recombinations with the chromosome of the Bacillus subtilisATCC 6633 strain.

At the same time, the plasposon pTnMod-RKm′ (Dennis and Zylstra, 1998.Plasposons: modular self-cloning minitransposon derivatives for rapidgenetic analysis of gram-negative bacterial genomes. Appl. Environ.Microbiol. 64, 2710-2715 [15]) was treated by the restriction enzymesNspl (Fermantas, Villebon sur Yvette, France; reference ER1471) and SacI(Fermentas, Villebon sur Yvette, France; reference ER1131) generating amixture of five fragments, including the fragment carrying rep(R6K) of451 pairs of bases (pb), the sequence of which has been isolated andpurified (Sambrook and Russell, 2001 [14]).

The fragments containing the sequences εpbp-P_(repU)-neo-εfenF andrep(R6K) were then ligatured together and (Sambrook and Russell, 2001[14]).

The sites of the restriction enzymes used were as follows:

-   -   Sphl: GCATGC (in position 1 of the sequence SEQ ID NO: 11)    -   Xbal: TCTAGA (in positions 743 and 2088 of the sequence SEQ ID        NO: 11) and    -   Sacl: GAGCTC (in position 2630 of the sequence SEQ ID NO: 11)

The cassettes εpbp, P_(repU)-neo and εfenF were composed in thefollowing manner:

-   -   cassette εpbp: from Sphl to Xbal (SEQ ID NO: 12),    -   cassette P_(repU)-neo: from Xbal to Xbal (SEQ ID NO: 13),    -   cassette εfenF: from Xbal to Sacl (SEQ ID NO: 14).

The ligature obtained was used to transform the stain of Escherichiacoli CC118(λpir) (Herrero, de Lorenzo and Timmis, 1990. Transposonvectors containing non-antibiotic resistance selection markers forcloning and stable chromosomal insertion of foreign genes ingram-negative bacteria. J. Bacteriol. 172: 6556-67 [16]) with aselection on a Luria-Bertani medium (or LB or Luria Broth medium)(Bertani, 2003, Lysogeny at mid-twentieth century: P1, P2 and otherexperimental systems. J. Bacteriol. 186, 595-600 [17]) containing 20μg/ml of neomycin. The plasmid obtained at E. coli(λpir) was calledpBG200 (3.1 kb)

FIG. 6 shows this construction schematically.

1.2 Protocol for Obtaining BBG116

The strain B. subtilis RFB102 (the strain derived from B. subtilis ATCC6633 obtained by insertion of the Pspac-comK cassette in amyE. Pspacdesignates the promoter issuing from the plasmid pA-spac (BacillusGenetic Stock Center, Columbus, Ohio, USA) inducible by IPTG, comKdesignates a gene essential for natural competence in Bacillus. It isassociated with a gene for resistance to spectinomycin (Pspac-comk-spc),which is integrated in the chromosome gene amyE. The strain RFB103 hasincreased ability for transformation by natural competence, which isinduced by IPTG (isopropyl-β-D-galactopyranoside). It was transformed bypBG200 previously treated by the plasmid amplification system TempliPhi(GE Healthcare), and then selected by means of resistance to neomycin,according to the protocol described in Dubnau, 1982 (Genetictransformation of Bacillus subtilis p 148-178. In D. Dubnau (Ed) Themolecular biology of the Bacilli, vol. I. Bacillus subtilis. AcademicPress, Inc. New York [18]).

Among the Nm-R clones, the insertion by double crossing-over of thecassette εpbp-P_(repU)-neo-εfenF in the chromosome of RFB102 wasverified by PCR using the primers PBP-FO2:

(SEQ ID NO: 1) AATAACGGACATGCCGAAGTG and FENF-REF2: (SEQ ID NO: 2)AATAGGCCGACCAAGACGTTC.

The overproduction of mycosubtilin in a Landy/MOPS medium at 22° C. wasverified in accordance with the operating method described in example 2below.

The strain B. subtilis BBG116 was thus obtained.

1.3 Protocol for Constructing the pBG144 Plasmid

A pBG212 plasmid of 6.5 kb dedicated to the insertional inactivation(“knock-out”) of the srfA operon of B. subtilis was constructed asfollows:

A EsrfAA (2.2 kb) cassette was generated by PCR using primers SRF-FOACAGGAATATGCTCAATCGAAG (SEQ ID NO: 3) and SRF-REV AAATTCGCTTCCAGGCTTCTG(SEQ ID NO: 4), from the genome DNA of B. subtilis subsp. subtilisstrain 168 (Accession NCBI PRJNA76) previously inserted in the plasmidpGEN=T Easy (Promega Corp, Charbonnières, France).

This amplicon was subsequently sub-cloned in the site EcoRI (Fermentas,Villebon sur Yvette, France; reference ER0271) of the vector pUC19 (NewEngland Biolabs, Ispwich, Mass., USA).

The EsrfAA cassette was then interrupted at the site Mfel (Fermentasreference ER0751) by insertion of the tet gene, previously generated byPCR using the primers TETP1 GTTGTATCGATGATGAAATACTGAATTTTAAACTTAG (SEQID NO: 5) and TETT1 TTTAATGGATCTAGAAGATTTGAATTCCTGTTAT (SEQ ID NO: 6),from the plasmid pBC16 (DSMZ GmbH, Brunswick, Germany), the originatorof Bacillus cereus (Accession: NC_001705.1).

The plasmid pBG144 was obtained by insertion, at the site BstEII(Fermentas, Villebon sur Yvette, France; reference ER0391) situated atthe end of the gene tet, of the gene cat previously generated by PCRusing the primers pC194cmfwd AGAAAGCAGACAGGTAACCCTCCTAA (SEQ ID NO: 7and pC194cmrev GCAGGTTAGTGACATTAGGTAACCGA (SEQ ID NO: 8) of the plasmidpC194 originating from Staphylococcus aureus (DSMZ GmbH, Brunswick,Germany, Accession: NC_002013.1).

FIG. 7 shows this construction schematically.

1.4 Protocol for Obtaining b. Subtilis BBG125Novel transformations of B. subtilis BBG116 with the plasmid pBG144previously linearised by AatII (Fermentas, Villebon sur Yvette, France;reference ER0991) in accordance with the protocol described in Dubnau,1982 (Genetic transformation of Bacillus subtilis p 148-178. In D. A.Dubnau (Ed) The molecular biology of the Bacilli, vol I. Bacillussubtilis. Academic Press, Inc. New York [18]).

Six Cm-R Tc-R clones were isolated on a gelosed LB medium containing theappropriate antibiotics. Checks by PCR were carried out using theprimers SRFAA5-FWD; AAGGAATCTCGCAATCATTTATCG (SEQ ID NO: 9) andSRFAA5REV; CTTGGTGTAAGCGGAATTTCTGTC (SEQ ID NO: 10). The non-productionof surfactin in a Landy/MOPS medium at 37° C. was verified in accordancewith the operating method described in example 2 below.

The mutant B. subtilis BBG125 was adopted as a monoproducing strain ofmycosubtilin.

The two strains B. subtilis BBG116 and BBG125 have haemolytic activitieson gelose containing 5% blood as well as antifungal activities on yeastsand moulds on a PDA medium.

These activities are more marked in the case of the strain B. subtilisBBG 116, because of the synergy between the surfactant produced and anoverproduction of mycosubtilins. These properties are in closerelationship with their ability to colonise the surface of the gelosedmedia, by virtue of the reduction in their surface tension.

The summarised characteristics of B. subtilis BBC125 and its parentalstrains are presented in table 1 below:

TABLE 1 Summarised characteristics of B. subtilis BBG125 and itsparental strains B. subtilis Parent strain Genotype Phenotype ATCC 6633— Wild Com⁻Myc⁺ Srf⁺ RFB102 ATTC 6633 amyE::Pspac- Com⁺⁺ Amy⁻ comK-spcMyc⁺ Srf⁺ Spc^(R) BBG116 RFB102 amyE::Pspac- Com⁺⁺ Amy⁻ comK-spc, Myc⁺Srf⁺ Spc^(R) myc::P_(repU)-neo Nm^(R) BBG125 BBG116 amyE::Pspac- Com⁺⁺Amy⁻ comK-spc, Myc⁺ Srf⁻ Spc^(R) myc::P_(repU)-neo, Nm^(R) Tc^(R) Cm^(R)srfAA::cat-tet

[Com: natural competence for transformation; Myc: production ofmycosubtilin; Srf: production of surfactin; Amy: amylolytic activity;Spc^(R), Nm^(R), Tc^(R), Cm^(R): resistances to spectinomycin,neomycin/kanamycin, tetracylin and chloramphenicol, respectively]

In comparison with the strain Bacillus subtilis ATCC 6633, the strainBacillus subtilis BBG125 produces a larger quantity of mycosubtilins anddoes not produce any surfactin.

Example 2: Preparation of the Culture Media 2.1 the Landy Medium

The composition of the Landy medium is as follows: glucose, 20 g/l;glutamic acid, 5 g/l, yeast extract, 1 g/l; K₂HPO₄, 1 g/l; MgSO₄, 0.5g/; KCl, 0.5 g/l; CuSO₄, 1.6 mg/1; Fe₂(SO₄)₃, 1.2 MG/l; MnSO₄, 0.4 mg/l.

2.2 Stock Solutions

In order to ensure reproducibility of the composition of the medium,sterile concentrated solutions were produced. A solution of 10× glucose(200 g/l) was sterilised by autoclaving at 121° C. for 20 minutes. Asolution of 4× glutamic acid (20 g/l) was adjusted to pH 8 with asolution of 5M KOH and was sterilised by filtration on a filter with aporosity of 0.2 μm. A 20× yeast extract solution (20 g/l) was sterilisedby autoclaving at 121° C. for 20 minutes. A solution of 40× n° 1minerals (K₂HPO₄, 40 g/l; MgSO₄, 20 g/l; KCl, 20 g/l) was acidified withconcentrated H₂SO₄ to total dissolution of the salts and were sterilisedby filtration on a 0.2 μm filer. A solution of 40× no 2 mineral salts(CuSO₄, 64 mg/l; Fe₂(SO₄)₃, 48 mg/l; MnSO₄, 16 mg/l) was acidified withconcentrated sulphuric acid to total dissolution of the salts and wassterilised by filtration on a filter with a porosity of 0.2 μm.

2.3 Production of One Litre of Landy Medium

The material used, apart from the pipettes, which are sterile and forsingle use, was previously sterilised by autoclaving at 121° C. for 20minutes, 250 ml of the glutamic acid solution was taken off in a sterilefashion and was then poured into an Erlenmeyer flask. 100 ml of theglucose solution was added thereto in a sterile fashion, and then 50 mlof the yeast extract solution and finally 25 ml of each of the mineralsolutions. The pH was adjusted to 7.0 by means of a sterile 5M KOHsolution. The volume was supplemented to 1 litre with sterile water.

2.4 Production of a Landy Medium Buffered with 200 mM of MOPS

A 20×MOPS buffer (2M) was produced by dissolving 20.93 g of3-[N-Morpholino]propanesulfonic acid (MOPS) (M1254, Sigma, St Louis,Mo., USA) in 50 ml of water. The solution was sterilised on a filterwith a porosity of 0.2 μm under a laminar-flow hood. To produce 1 litreof Landy medium buffered to 100 mM with MOPS, 50 ml of 20×MOPS was addedto the mixture produced in example 2.3.

Example 3: Culture of B. subtilis BBG125 in Erlenmeyer Flasks 3.1Preparation of a Strain Collection

A screw-type tube containing 5 ml of modified E medium (the Clark Emedium is modified by reducing the glucose concentration from 40 to 20g/l. The composition of the medium is as follows: KH₂PO₄, 2.7 g/l;K₂HPO₄, 18.9 g/l; yeast extract, 0.5 g/l; glucose, 20 g/l; EDTA, 0.05g/l; MgSO₄, 0.61 g/l; MnSO₄, 0.056 g/l; NaCl, 0.1 g/l; CaCl₂, 0.012 g/l;ZnZO₄, 0.018 g/l; FeSO₄, 0.018 g/l; CuSO₄, 0.002 g/l; Na₂MoO₄, 0.001g/l; H₃BO₃, 0.001 g/l; Na₂SO₃, 0.001 g/l; NiCl₂, 0.0037 g/l; NH₄NO₃, 4g/l; MgSO₄, 1 g/l. The pH of the solution is adjusted to 6.5 with a 10%HCl solution) was inoculated with a colony of the primary straincollection of B. subtilis BBG125 and set to incubate at 30° C. for 24hours under a stirring of 300 rotations per minute (rpm). The solutionwas then homogenised by vortex. A volume of 1.5 ml of the cultureobtained previously was added to 48.5 ml of modified E medium containedin a 500 ml Erlenmeyer flask. The whole was set to incubate at 30° C.for 12 to 24 hours under stirring of 120 rpm. This first preculture P1was duplicated.

The culture was then homogenised by vortex, and the DO_(600 nm) was thenmeasured with a spectrophotometer (SECOMAN Prim, SECOMAN, Domont,France) until the B. subtilis BBG125 strain was at the start/middle ofan exponential growth phase.

A P2 preculture was inoculated with 0.5 ml of the culture of the bestflask P1 and was duplicated. The 500 ml Erlenmeyer flasks contained, asthe final volume, 50 ml of modified E medium and were incubated at 30°C. under 120 rpm stirring. The growth was stopped when the DO_(600 nm)indicated that the culture was at the start/middle of an exponentialgrowth phase (1<DO_(600 nm)<5). The purity and quality of P2 werechecked, by observation with a microscope and by seeding with anutritive Luria-Bertani gelose (Tryptone, 10 g/l; yeast extract, 5 g/l;NaCl, 10 g/l; pH 7.2 and a Mossel gelose (meat extract, 1 g/l; peptone,10 g/l; D-mannitol, 10 g/l; NaCl, 10 g/l; phenol red, 0.025 g/l, agar,12 g/l; egg yolk, 10 ml/l; polymyxin, 5 ml/l; pH 7.1)), more specific ofthe bacilli, complemented with spectinomycin at 100 μg/ml. The disheswere set to incubate at 30° C. for 24 hours.

It should be noted that B. subtilis produces colonies with irregularshapes (the contours are undulating and may exhibit filaments), with acreamy consistency, the diameter of which is between 2 and 4 mm. In oldcultures, the colonies adopt a dry, rough appearance and becomeencrusted in the gelose.

Finally, a 2 litre flask containing 200 ml of modified E medium definedabove was inoculated at 5% with the best flask of P2. This flask wasincubated at 30° C. under stirring of 120 rpm and the growth was stoppedwhen the DO_(600 nm) indicated that the culture is at the start/middleof an exponential growth phase (1<DO_(600 nm)<5). The quality and purityof the culture were checked as indicated for P2. The culture wascentrifuged at 2000 g for 10 minutes at 25° C. The residues were washedin sterile physiological water and then the suspensions were centrifugedat 2000 g for 10 min at 25° C. The residues were taken up in a volume ofE medium without antibiotic, so as to obtain a final DO_(600 nm) of 25per tube. The suspension was distributed in cryotubes at the rate of 0.9ml of culture and 0.6 ml of glycerol. The tubes were homogenised byvortex and stored at −80° C. The E medium was supplemented withspectinomycin at 100 μg/ml.

3.2 Preparation of an Inoculum

The inoculum was prepared from the strain collection containing cellskept at −80° C. in 40% glycerol. A tube containing 5 ml of modified Emedium defined below was adjusted to pH 7.0 with a 10% HCl solution(v/v) and was inoculated with 0.5 ml of bacterial suspension of thestrain collection. The whole was set to incubate at 30° C. for 10 to 14hours under stirring of 300 rpm. The tube was then homogenised by vortexand the DO_(600 nm) was measured. A preculture P1 is then produced in afinal volume of 50 ml of modified E medium at pH 7.0 contained in a 500ml Erlenmeyer flask. The whole was set to incubate at 30° C. understirring of 140 rpm, the preculture was stopped when the strain wassituated at the start/middle of an exponential growth phase(1<DO_(600 nm)<5). This first preculture P1 was duplicated.

A second preculture P2 was produced in the same way as the preculture P1and this was inoculated from the first flask of P1 and was duplicated.The volume necessary for starting the cultures in flasks was thencentrifuged at 2000 g for 10 min at 25° C. The residue was put back insuspension in 10 ml of sterile physiological water. The suspensionobtained was centrifuged once again at 2000 g for 10 min. The residuewas finally taken up in 10 ml of sterile physiological water. Thesuspension was then ready for inoculation.

3.3 Cultures in Erlenmeyer Flasks

The experiments lasted for a minimum of 72 hours and several sampleswere taken from these cultures. The initial DO_(600 nm) is between 0.1and 0.4. The volume of the Erlenmeyer flasks was 500 ml and the volumeof the nutritive medium was 100 ml. The following measurements wereperformed on the samples taken in a sterile fashion under thelaminar-flow hood: a check on the purity by isolation on nutritivegelose and Mossel gelose+spectinomycin (100 μg/ml), a measurement of theoptical density at 600 nm, a measurement of the pH, a measurement of thedry weight and the taking off of the culture supernatant forquantitative analysis of the lipopeptides by HPLC: 3 ml of culture iscentrifuged for 10 minutes at 10,000 g at 4° C. and the culturesupernatant was stored at −20° C.

Example 4: Purification and Analysis of the Lipopeptides 4.1Purification of the Lipopeptides

The lipopeptides were extracted on cartridges of 1 g of Maxi-clean C18gel (Grace Davison-Alltech, Deerfield, Ill., USA).

A cartridge of 1 g of ODS was conditioned with 100% methanol, with 20 mlat the first pass and then 8 ml. The cartridge was then rinsed with 8 mlof milli-Q water (Millipore). 1 ml of culture supernatant with a pH of6.5±0.1 was then loaded onto the column. The cartridge was then washedwith 8 ml of milli-Q water. After drying of the cartridge with 20 ml ofair, the lipopeptides were eluted with 4 ml of 100% methanol. The eluatewas dried by means of a vacuum concentrator. The sample was subsequentlytaken up in 200 μl of 100% methanol at 4° C. to enable HPLC analysis.

4.2 Analyses by High Performance Liquid Chromatography (HPLC)

The sample was analysed by means of a complete HPLC system, make Waters(Online Degasser, 717 Autosampler, 660S Controller, 626 Pump, 2996PhotoDiodeArray) (Waters SAS, Guyancourt, France) using a C18 column (5μm, 250×2.5 mm, VYDAC 218 TP). Two analyses were carried out.

The first analysis was that of the mycosubtilins: 10 μl of purifiedsample was injected and compared with an iturin A standard at 500 mg/min(11774, Sigma-Aldrich, St Louis, Mo., USA) with a flow rate of 0.6ml/min. The elution was carried out in isocractic mode using a 60/40/0.1(v/v/v) water/acetonitrile/trifluoroacetic acid solvent.

The second analysis was that of the surfactins. 10 μl of purified samplewas injected compared with a surfactin standard at 500 mg/l (S3523,Sigma-Aldrich, St Louis, Mo., USA) with a flow rate of 0.6 ml/min. Theelution was carried out in isocratic mode using a 20/80/0.1 (v/v/v)water/acetonitrile/trifluoroacetic acid solvent.

The retention time and the second drift of the spectrum between 200 and400 nm of each peak (diode array, PDA 2996, Waters) were analysedautomatically by means of Millennium software for identifying the elutedmolecules.

4.3 Analyses by Semi-Preparative HPLC

The sample was prepared by applying the purification protocol describedin example 4.1 above, using cartridges of Maxi-clean C18 gel (GraceDavison-Alltech, Deerfield, Ill., USA) of 10 g. The use of 10 gcartridges made it possible to load 10 ml of culture supernatant insteadof 1 ml as before. All the volumes were multiplied by a factor of 10,except for the volumes of methanol. The minimum volume of methanol usedfor conditioning the cartridge and then eluting the lipopeptides was 10ml. The sample was loaded manually (100 μl) into the injection system ofthe semi-preparative HPLC, make Waters (660 Controller, 626 Pump, 486Absorbance Detector). The column used was a C18 (5 μm, 300×10 mm, ACE).The elution was carried out at a rate of 3 ml/min in accordance with thegradient presented in table 2 below:

TABLE 2 Elution according to the respective concentrations of buffers Aand B Time (min Buffer A (%) Buffer B (%) 0 65 35 4 65 35 54 50 50 60 0100 61 65 35 65 65 35

The solvents used are as follows: solvent A composed of water andtrifluoroacetic acid, 99.9/0.1 (v/v) and solvent B composed ofacetonitrile and trifluoroacetic acid, 99.9/0.1 (v/v).

4.4 Analyses by MALDI-TOF Mass Spectrometry

The analyses by MALDI-TOF mass spectrometry (Bruker Ultaflex) werecarried out according to requirements using: either culturesupernatants, or samples purified on ODS cartridge (GraceDavison-Altech, Deerfield, Ill. USA) or samples purified on ODScartridge and by semi-preparative HPLC. A TA buffer was prepared byproducing a 33/67/0.1 (v/v/v) CH₃CH/water/trifluoroactic acid mixture. ACHCA buffer is a saturated solution of alpha-cyano-4-hydroxycinnamicacid in TA buffer. This buffer was prepared by recovering thesupernatant after centrifugation of the alpha-cyano-4-hydroxycinnamicacid/TA buffer. The samples to be analysed were prepared by mixing 1 μlsample with 9 μl of HCA buffer. The solution of sample deposited byMALDI-TOF analysis represented a volume of 0.5 μl. Drying was carriedout in open air. The masses were calibrated with a mixture of standardpeptides.

The calculated masses of the ions [M+H]⁺, [M+Na]⁺, [M+K]⁺ of the varioushomologues of mycosubtilins and surfactins obtained are specified intable 3 below:

TABLE 3 Calculated masses of the [M + H]⁺, [M + Na]⁺, [M + K]⁺ ions ofthe various homologues of mycosubtilins and surfactins MassesLipopeptides [M + H]⁺ [M + Na]⁺ [M + K]⁺ Surfactin C₁₃ 1008.66 1030.641046.61 Surfactin C₁₄ 1022.67 1044.66 1060.63 Surfactin C₁₅ 1036.691058.67 1074.65 Mycosubtilin C₁₅ 1057.57 1079.55 1095.52 MycosubtilinC₁₆ 1071.58 1093.56 1109.54 Mycosubtilin C₁₇ 1085.60 1107.58 1123.55

The HPLC analysis revealed 10 peaks for which the molecular masses ofthe molecules detected are presented in table 4 below:

TABLE 4 Masses of the 10 peaks detected by HPLC analysis Peak No MassPeak no Mass 1 1056 6 1098 2 1084 7 1098 3 1084 8 1084 4 1070 9 1084 51070 10 1098

4.5 Analyses of the Structure of the Novel Mycosubtilins by MS-MS

In order to precisely determine the structure of the various forms ofmycosubtilin produced by the BBG125 strain, an analysis of the purifiedsamples was carried out by tandem mass spectrometry (MS-MS) withionisation of the electrospray type (Ion Trap Finnigan MAT LCQ) indirect infusion mode after starting the peptide cycle by means of atreatment with N-bromosuccinimide in a concentration equivalent tomycosubtilin in a 70% acetic acid solution.

A first analysis was carried out on the purified C17 anteisomycosubtilin (peak 8). The spectrum obtained (MS1) is complicated by thetwo isotopes of Br. The spectrum MS2 is complicated by the presence offragments with 1, 2 or 3 —NH₃ groups missing, owing to the presence ofthe Asn and Gln amino acids. The MS spectrum of the starting productgives the peaks 1085 [M+H]⁺, 1107[M+Na]⁺ and 1123 [M+K]⁺ correspondingclearly to C17 mycosubtilin.

Because of the presence of the isotopes 79BR and 81Br in fairly similarquantities, a distribution of peaks around 1260 is observed that doindeed correspond to the expected drift. For example, the peak at 1257.4corresponds to [M+H]⁺ with two 79Br, the peak at 1259.4 corresponds to[M+H]⁺ with 79Br and 81Br or to [M+H]⁺ with two 13C and two 79Br, etc.

The order of the increasing masses obtained from the fragmentation ofthe ion at 1257.4 is set out in table 5 below:

TABLE 5 Increasing masses obtained from the fragmentation of the ion at1257.4 m/z Ion fragment 365 Vn-NH₃ 382.4 vN 391.6 b4-H₂O—NH₃ 432 y2-NH₃434.4 SNv-H₂O—NH₃ 452.3 Snv-NH₃ 462.3 NvN—2NH₃ 496.4 NvN 506.2 b5-2NH₃531.3 SNvN-H₂O—2NH₃ or PSNV-H₂O—NH₃ 549.4 SNvN—2NH₃ or PSNv-NH₃ 566.3PSNv or SNvN—NH₃ 583.4 SNvN 700 Y3—NH₃ 717.1 y3 814.0 y4-NH₃ 831.1 y4918 y5 664 y6-3NH₃ 981.1 y6-2NH₃ 998.1 y6-NH₃ 1015.1 y6 1091.9 Y7—3NH₃1109.1 Y7—2NH₃ 1126.0 y7-NH₃ 1143 y7 1420.0 M-H₂O — —

In the above table, “m/z” means mass to charge ratio.

4.6 MS-MS Analysis of the Peak at 1274.4 Contained in Peak 6

Isotope mass before treatment: M=1098.6

Isotope mass after treatment, with 79BR2: M=1270.4

Fragment y identical to C₁₇ anteiso mycosubtilin.

Fragments b greater than 14 with respect to the fragments b of C₁₇anteiso mycosubtilin.

This means that very probably one of the first two amino acids in theopen sequence (N or Q of NQPSNvNw) has been modified.

4.7 MS/MS Analysis of the Peak at 1274.4 Contained in Peak 10

Isotope mass before treatment: M=1098.6

Isotope mass after treatment, with 79Br1: M=1270.4

Fragments y identical to mycosubtilin anteiso C₁₇ but with 14 more,including the intense y6 peak at 1029 instead of 1015 for all the othersamples.

Fragments b less than b6 identical to anteiso C₁₇ mycosubtilin.

Fragments b greater than or equal to b6 identical to mycosubtilin butwith 14 more.

These results show us that the molecule would be mycosubtilin with a C₁₈rather than C₁₇ fatty acid.

On the basis of the order of elution of the various peaks, we deducefrom this the existence of 5 novel forms of mycosubtilin: the formsGln3, iso C₁₆, nC16, anteiso C₁₇, iso C₁₇ and a C₁₈ form. Thecorrespondence of these forms with the ten peaks with the molecularmasses of the molecules detected by the HPLC analysis of example 4.5 ispresented in table 6 below:

TABLE 6 Correspondence between the masses of the 10 peaks detected byHPLC analysis and the mycosubtilin forms Peak no Mass Form 1 1056 C₁₆ 21084 isoC₁₆ GLN 3 1084 _(n)C₁₆ GLN 4 1070 isoC₁₆ 5 1070 _(n)C₁₆ 6 1098anteisoIC₁₇ GLN 7 1098 isoC₁₇ GLN 8 1084 anteisoC₁₇ 9 1084 isoC₁₇ 101098 C₁₈

The formula of this novel C₁₈ mycosubtilin is as follows:

The formula of this novel C₁₇ Gln3 micosubtilin is as follows:

Example 5: Biological Activity of Various Isoforms of Mycosubtilin (MS)Vis-à-Vis Various Microorganisms

Tests on antifungal activities were carried out by successive dilutionsof the C₁₈ isoform (C₁₈ MS) in a liquid medium according to the protocoldescribed in Besson et al. (Besson et al. 1979. Antifungal activity uponSaccharomyces cerevisiae of iturin A, mycosubtilin, bacillomycin L andof their derivatives; inhibition of this antifungal activity by lipidantagonists. J. Antiobiot. (Tokyo) 32, 828-833) [19]. Cultures in96-well microplates were carried out in a rich medium: glucose, 40 g/l;peptone, 10 g/l; yeast extract, 2 g/l; pH=7.2. Seeding of Saccharomycescerevisiae was carried out at a DO_(600 nm) of 0.55 and the absorbancewas read after 24 hours and the minimum inhibiting concentration (MIC)was then determined.

This experiment was also carried out with the following isoforms ofmycosubtilin (MS): MS iso-C₁₆, MS n-C₁₆, MS anteiso-C17, MS iso-C₁₇. Itwas also carried out with a composition of mycosubtilins (MS comp.)comprising, as a percentage with respect to the weight of thecomposition, 26% of MS iso-C₁₆, 1% MS Gln3 C₁₇, 2% MS n-C₁₆, 44% MSanteiso-C₁₇, 23% MS iso-C₁₇ and 1% MS C₁₈.

This experiment was also carried out, for each of the aforementionedisoforms of mycosubtilins and composition on the followingmicroorganisms: Botrytis cinerea, Aspergillus niger, Sclerotiniasclerotium, Candida albicans.

The MICs obtained for each of these experiments are presented in table 7below:

TABLE 7 MIC of various isoforms of mycosubtilin and a compositionvis-à-vis various microorganisms MIC (μM) Micro- MS MS MS MS MSorganisms iso-C₁₆ n-C₁₆ anteiso-C₁₇ iso-C₁₇ comp. B. cinerea 32 16 8 168 A. niger 32 16 8 16 8 C. albicans >32 8 32 16 8

These results show that the composition MS comp. has effects equivalent,or even superior, to the mycosubtilins currently used.

Example 6: Implementation of an Integrated Method for Producing,Extracting and Concentrating the Lipopeptides Produced by B. subtilis onAir/Liquid Membrane Contactor

The description of this example refers to FIGS. 1 to 5.

In this example:

-   -   the pumps P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13        and P14 were Masterflex L/S peristaltic pumps compact drive        model (Cole Parmer, Vernon Hills, Ill., U.S.A.),    -   the pump P11 was of the N820.3 FT.18 type (KNF Neuberger        Laboport, Freiburg, Germany),    -   the valves V1, V4 and V5 were stop valves made from PTFE        (W3250Y, Thermo Fisher Scientific, Roskilde, Germany),    -   the valves V2, V3, V6 and V7 were three-way stop valves made        from PTFE (W3250Z, Thermo Fisher Scientific, Roskilde, Germany),    -   the tanks tank 2 and tank 4 were Nalgene tanks made from        high-density polypropylene with a useful volume of 4 litres        (2125-4000 Heavy Duty Bottles, Nalgene, Thermo Fisher        Scientific, Roskilde, Germany),    -   the tanks tank 1, tank 3, tank 5, tank 6 and tank 7 were Nalgene        tanks made from high-density polypropylene with a useful volume        of 10 or 20 litres (2250 Autoclavable Carboys, Nalgene, Thermo        Fisher Scientific, Roskilde, Germany).    -   the scales B1, B2, B3, B4, B5, B6 and B7 were of the CKW-55        type; Ohaus Corporation, Pine Brook, N.J., U.S.A.),    -   the strain of Bacillus subtilis was the strain B. subtilis        BBG125.

6.1 Environmental Conditions and Sensors Used for the Culture

Unless indicated to the contrary, the pH was regulated to 7+/−0.1 bymeans of the controlled addition, respectively by the pumps P2 and P3,of solutions of 0.66M H₃PO₄ or 3M NaOH sterilised previously byautoclaving at 121° C. for 20 minutes.

A pH electrode was calibrated before autoclaving of the tank usingcommercial solutions buffered to pH 4.0 and pH 7.0 and stored at 4° C.The process was conducted at 22°+/−0.1° C. by means of an Alpha Laval 1m² tubular heat exchanger (104878, Alpha Laval Corporate AB, Lund,Sweden). The concentration of dissolved oxygen pO₂ was measured by meansof an oxygen sensor (Mettler Toledo, Viroflay, France). The electrolyteof the oxygen sensor was renewed at each experiment. The oxygen sensorwas calibrated after autoclaving of the tank when the culture mediumreached the set temperature and pH of the experiment. The 0% of pO₂ wasobtained by connecting the cable of the sensor to earth and the 100% pO₂by saturating the medium with air (1000 rpm and 1 vvm).

The aeration rate (Fe) was fixed at 0.25 volumes of air per volume ofliquid per minute (vvm), that is to say 0.75 litres/min for 3 litres ofLandy medium (example 2.1). The incoming air was filtered through a 0.2μm sterilising filter.

The software used for controlling the process and acquiring the data wasAFS Biocommand (New Brunswick Scientific, Edison, N.J., U.S.A.). Thepurity of the culture was checked after 48 hours and at the end ofculture. Culture samples of 10 ml were regularly taken and centrifuged,the optical density and the dry weight were determined, and thesupernatant was stored before analysis. The incoming and outgoing gaseswere analysed in order to obtain data on the respiration of themicroorganism. A paramagnetic sensor made it possible to analyse thequantity of oxygen and an infrared sensor that of the carbon dioxide(Xentra 4400; Servomex Company Inc., Sugar Land, Tex., U.S.A.). Theanalyser was integrated in a multiplexed device that afforded asequential analysis on six channels, drying of the gases on Naflonmembrane (Permapur, Saint-Leonard, Quebec) and automatic calibration.

6.2 The Air/Liquid Membrane Contactor

The air/liquid membrane contactor M1 used in this example is supplied byGE-Healthcare, reference CFP-6-D-45 (GE-Healthcare Europe GmbH, Munich,Germany). It consists of an external module comprising two compartmentsC1 and C2. In compartment C1, a sterile gas circulates containingoxygen. In compartment C2, the culture medium containing the inoculumcirculates at a rate of 24 litres/h/m² of membrane imposed by the pumpP4.

The membrane M1 has a surface area of 2.5 m² and is sterilised beforeuse by autoclaving at 121° C. for 20 minutes (this criterion is notexhaustive). The membrane consists of a set of hollow polyethersulfonefibres having a porosity of 0.65 μm.

6.3. Device for Continuous Culture of Bacillus subtilis: Coupling of aSystem for Extraction/Concentration of the Biosurfactants by Air/LiquidMembrane Contactor6.3.1. Coupling of a System for Extraction/Concentration of theLipopeptides with the Air/Liquid Membrane Contactor.

The device used for the continuous culture of B. subtilis comprises anair/liquid membrane contactor M1 made from hollow fibres in which B.subtilis BBG125 has been cultivated in a Landy medium. B. subtilisBBG125 immobilised on the surface of the membrane M1 degrades thissubstrate by excreting biosurfactants. The device also comprises meansfor supplying and drawing off a given flow rate of said substratecontinuously in the production device comprising the membrane M1.

The membrane M1 provides, in a sterile environment, the oxygen necessaryfor the growth of the microorganism in the culture medium, by means ofthe diffusion of oxygen through its pores.

A peristaltic pump P1 continuously supplies the membrane M1, at a rateF1, with fresh Landy medium stored in a tank 1, and likewise aperistaltic pump P5 continuously draws off the culture medium from theproduction device comprising the membrane M1 described above at a rateF2.

The inoculum and the fermentation conditions remain equivalent to thosedescribed previously. In order to keep the environment sterile, all theconstituents of the device and the various membranes used weresterilised at 121° C. for 20 minutes.

One feature of this device stems from the fact that it is possible torecycle or not, in the production device comprising the membrane M1, allthe microorganisms cells after having removed from them the residues oforganic matter and biosurfactants, which makes it possible to obtain ahigh rate of growth of the microorganisms in the production device.

Moreover, this device enables oxygen to be diffused in the culturemedium without the formation of bubbles or foam.

6.3.2. Extraction of the Lipopeptides and Recycling of the Cells byMicrofiltration

The device described previously is therefore characterised by the factthat it comprises tangential microfiltration and ultrafiltration means,situated at the discharge from the production device, which separate theliquid into several fractions.

A first microfiltration step was performed on a membrane M2 made fromhollow polyethersulfone fibres with a pore size of 0.2 μm (GEHealthcare) with a surface area of 0.4 m², in which the culture mediumcontaining the cells circulates, by means of the volumetric pump P4described above, in the compartment C3 of the membrane M2 referred to asthe residue. Under the effect of a volumetric pump P5, the medium passesby tangential filtration through the pores of the membrane M2 to thecompartment C4 of the membrane M2. By this means, the culture mediumcontaining the biosurfactants has the cells removed and is extracted andthen collected in a tank 2 stirred by blades driven by a motor B or bymagnetic agitation (magnetic agitator, W10512, Thermo Fisher Scientific,Roskilde, Germany) at a speed of 160 rpm.

The culture medium of the production device comprising the membrane M1is taken off continuously to the tank 2. In order to compensate for thisdrawing off and to keep a constant volume in the membrane M1, the pumpP1 supplies the bioreactor with new medium contained in tank 1.

Moreover, tank 2 and tank 3 are placed respectively on scales B2 and B3.It is the latter that control the output of the peristaltic pump P1(CKW-55; Ohaus Corporation, Pine Brook, U.S.A.), making it possible tomaintain a constant volume inside the membrane M1 and thereby obtainingF1=F2. In each of the experiments carried out, the degree of dilution ischanged after the passage of at least four air/liquid membrane contactorvolumes comprising the membrane M1.

The outputs of the pumps P1 and P5 are equal and adjusted so as toobtain, in the device comprising the membrane M1, a degree of dilutionof 0.1 h⁻¹, that is to say an hourly flow rate equal to 0.1 times thevolume of the aqueous phase contained in the production devicecomprising the membrane M1.

A variant of this method has also been implemented. It consists ofrecycling or not the cells inside the membrane M1. It is then an opencontinuous mode presented in broken lines in FIGS. 3 and 4. In the caseof non-recycling by the set of valves V6, V7 and V8, the culture mediumcan be pumped by the pump P13 from the membrane M1 and collected in thetank 7. A drain valve makes it possible to eliminate the cellconcentrate intermittently. In this case, the step of microfiltration bythe membrane M1 is performed directly on the medium contained in thetank 7 and the cells are then concentrated in the tank 7 rather than inthe production device comprising the membrane M1.

6.3.3. Concentration of the Lipopeptides by Ultrafiltration

The filtrate contained in the tank 2 is finally driven, by means of apump P6, into the compartment C5 of a stainless steel tangentialultrafiltration system (Sartocon 2 plus, 17546---202, Sartorius,Göttingen, Germany), comprising an ultrafiltration membrane M3 with acutoff threshold of 10 kDa made from regenerated cellulose (HydrosartUltrafilter, 3021443930E-BSW, Sartorius, Göttingen, Germany). Above thecritical micell concentration, the biosurfactants are concentrated inthe compartment C5, referred to a residue, and thus return to the tank2. The culture medium is extracted, under the control of a pump P7, at arate equivalent to that of the pump P5, to a compartment C6, calledultrafiltrate, and collected in a tank 3. The flow rate will depend onthe flow rate imposed by the pump P7 and regulated by means of theinformation collected by the various scales B2, B3 and B4.

6.4. Purification of the Lipopeptides 6.4.1. Purification byUltrafiltration/Diafiltration

The purification of the lipopeptides presented in this example is basedon the concatenation of ultrafiltration steps through a stainless steeltangential ultrafiltration system (Sartocon 2 plus, 17546---202,Sartorius, Göttingen, Germany), comprising an ultrafiltration membraneM4 with a cutoff threshold of 10 kDa made from regenerated cellulose(Hydrosart Ultrafilter, 3021443930E-BSW, Sartorius, Göttingen, Germany).This step aims to eliminate from the culture broth a major part of theresidual substances such as glucose, glutamate and the various primarymetabolites. The formation of micells and micell complexes by themycosubtilin, when it is situated above its CMC, makes it possible toretain it and therefore concentrate it in the residue, by virtue of theuse of the ultrafiltration membrane M4.

This step is performed at 25° C. and at a pressure of 0.5 bar. Thispurification step is performed on the concentrated lipopeptides in thetank 2. After opening of the valve V1, the pump P8 drives theconcentrate to the tank 4 (Nalgene, made from high-density polypropylenewith a useful volume of 4 litres (2125-4000 Heavy Duty Bottles, Nalgene,Thermo Fisher Scientific, Roskilde, Germany)) stirred by blades drivenby a motor C or by magnetic agitation (magnetic agitator, W10512, ThermoFisher Scientific, Roskilde, Germany) identical to the motor B.

The following steps are performed sequentially:

-   -   Ultrafiltration: The concentrate is transferred into the tank 4,        under the control of a Masterflex L/S peristaltic pump P8        compact drive model (Cole Parmer, Vernon Hills, Ill., U.S.A.),        and then purified on the membrane M4, under the control of a        Masterflex L/S peristaltic pump P9 compact drive model (Cole        Parmer, Vernon Hills, Ill., U.S.A.) and collected in the tank 4.        The Masterflex L/S peristaltic pump P10 compact drive model        (Cole Parmer, Vernon Hills, Ill., U.S.A.) makes it possible to        pass the remainder of the constituents of the medium to the        compartment C8 and the tank 5 (Nalgene made from high-density        polypropylene with a useful volume of 10 or 20 litres (2250        Autoclavable Carboys, Nalgene, Thermo Fisher Scientific,        Roskilde, Germany)). This process is continued until the volume        contained in the tank 4 is reduced to 10% of the volume        initially in the tank 4.    -   Diafiltration: This step dilutes the culture broth in order to        facilitate the passage of the residual substances through the        ultrafiltration membrane M4. The water is then added to the tank        4 by opening the valve V2 until the volume in the tank 4 regains        its original level. There follows an ultrafiltration step as        described above. This diafiltration step is performed four times        in succession.    -   Ultrafiltration in the presence of methanol (MeOH): Following        the diafiltration steps, MeOH is added from the tank 6 (Nalgene        made from high-density polypropylene with a useful volume of 10        or 20 litres (2250 Autoclavable Carboys, Nalgene, Thermo Fisher        Scientific, Roskilde, Germany) to the tank 4 via the Masterflex        L/S peristaltic pump P12 compact drive model (Cole Parmer,        Vernon Hills, Ill., U.S.A.) and the valve V2. This addition of        MeOH is controlled by the scales B4, B5 and B6 so that the        solution present at this time in the tank 4 contains 70% MeOH        (v/v). There follows an ultrafiltration step as described above.        This step will destroy the micells and pass the mycosubtilin        monomers through the pores of the membrane M4.

After filtration, a solution consisting of mycosubtilin and 70% methanolis collected on the ultrafiltrate side, but this time the ultrafiltrateis collected in the vessel of a Rotavapor VV2000 evaporator (Evapo)(Heidolph Instruments GmbH & Co., Schwabach, Germany) by means of theset of valves V3 and V4.

At the end of each step, samples are taken on the filtrate side and theresidue side. Thus the balance of the purification can be established.This makes it possible to determine the mycosubtilin losses caused bythe ultrafiltration and diafiltration steps, by calculating the ratio ofthe quantity of concentrated mycosubtilin obtained after ultrafiltrationto the quantity of mycosubtilin initially present in the tank 4. Theyield of these steps is greater than 70%.

6.4.2. Concentration of the Lipopeptides by Evaporation

The evaporator (Evapo) concentrates the mycosubtilins by removing allthe methanol and some of the water. This evaporation takes place at aresidual pressure of 50 mbar imposed by the vacuum pump P11, type N820.3FT.18 (KNF Neuberger Laboport, Freiburg, Germany) and at 50° C. Duringthis step, the methanol is evaporated and its vapours are condensed bymeans of the condenser, and is recycled in the tank 6.

6.4.3. Freeze Drying of the Lipopeptides

An optional freeze drying step was added to this method in order toimprove the preservation of the product. The freeze drying of thelipopeptides is performed directly using a concentrated solution Xissuing from the evaporation and recovered by the valve V5. This isfirst of all frozen at −20° C. and then freeze dried by means of a HetoPower Dry PL 9000 freeze dryer (Jouan Nordic, Allerod, Denmark), inaccordance with the following steps: 1 hour at −30° C.; 5 hours at −10°C.; 5 hours at 0° C.; 5 hours at −20° C.; 5 hours at 35° C. The freezedrying was carried out at a residual pressure of 15 mbar.

Example 7: Washing of the Membranes and Recovery of the Lipopeptides

Because of the affinity of the lipopeptides for the interfaces, theprotocol for washing the membranes was studied and optimised. It takesaccount of the nature of the membranes and is in agreement with therecent work published on this subject (Chen, Chen and Juang, 2007.Separation of surfactin from fermentation broths by acid precipitationand two-stage dead-end ultrafiltration processes. J. Membr. Sci. 299,114-121 [20]; Chen, Chen, and Juang, 2008. Flux decline and membranecleaning in cross-flow ultrafiltration of treated fermentation brothsfor surfactin recovery. Sep. Purif. Technol. 62, 47-55 [21]). Thesewashings denature neither the lipopeptides nor the membranes. The use ofsolvents is proscribed although some, such as methanol, are veryeffective for detaching the lipopeptides. Tests for sensitivity to pHdetermined that a pH=10 is the limit of degradability of thelipopeptides. The washings are performed under stirring and fermentationconditions. The protocol is implemented in seven water-based washingsteps.

-   -   Two washings were performed with 3 litres of distilled water at        30° C. for 30 minutes. These detached the slightly immobilised        biomass.    -   The second washing with distilled water at 30° C. made it        possible to measure the oxygen transfer coefficient.    -   Two washings were performed with 3 litres of 0.1 M NaOH at        50° C. for 1 hour. These detached the highly immobilised biomass        and desorbed the majority of the lipopeptides.    -   The membrane was then regenerated with a 0.5 M solution of NaOH        at 50° C. for 1 hour then with a 100 ppm solution of NaOCl at        50° C. for 1 hour, and was then cleaned with distilled water at        25° C. until neutrality was achieved in the membrane.

The whole of the aforementioned method was also implemented byduplicating each membrane, so that it can be washed and regeneratedsequentially. This made it possible to implement the method withoutdiscontinuing. In order to implement this alternative method, tanks (notshown) containing the 0.1 and 0.5 M soda solution were added to thedevice.

Example 8: Quantification of the Biosurfactants Produced

In this example, several tests were performed to produce biosurfactantsby B. subtilis by fermentation of glucose at a concentration of 20 g/l,in a production device containing 3 litres of culture medium and a totalsurface area of air/liquid membrane contactor of 2.5 m², conforming tothe air/liquid membrane contactor described in example 6. Each of theexperiments was repeated twice. Only the average of these doublets ispresented below. The standard deviation is between 5% and 15%.

The experimentation conditions were adjusted as follows:

-   -   the pH was maintained at 7,    -   the air flow in the air/liquid membrane contactor was 1 vvm,    -   the culture medium volume was 3 litres circulating in the fibres        of said membrane at a speed of 0.021 m/s,    -   the pressure of the whole of the system was atmospheric        pressure, except at the air inlet, this may be slightly above        atmospheric pressure at 0.4 bar,    -   the oxygenation conditions of the culture medium were fixed with        a volumetric oxygen transfer coefficient of around 40 h⁻¹.

Chromatographic analysis by HPLC quantified the substrates andbiosurfactants in the various tanks.

Analysis of the oxygen consumed made it possible to determine thequantity of cells immobilised on the membrane.

The following formula was used to determine the biomass immobilised onthe membrane over time in (g m⁻²) considering that the free andimmobilised cells have different specific oxygen consumption rates,oxygen being more accessible for the cells immobilised on the membranethan for those in suspension:

biomass immobilized on the membrane at a given time in (gm⁻²)=(OUR−(X*OUR_(spe cl)))*V/(OUR_(spe ci) *a)

in which:OUR=oxygen consumption rateOUR_(spe cl)=specific oxygen consumption rate of the free cellsOUR_(spe cl)=specific oxygen consumption rate of the immobilised cellsV=reaction volumeX=concentration of free biomassa=surface area of the membrane

The steps of washing the membranes described in these examples wereperformed in accordance with the method described in example 7.

8.1 Production of Mycosubtilin by B. subtilis BBG125, Batch Mode

In this example, the B. subtilis BBG125 strain was cultivated in batchesat 30° C. for 48 hours in an air/liquid membrane contactor M1.

8.1.1 Analysis of the Biomass Produced.

In this method two types of biomass were observed, one in freesuspension in the culture medium and the other immobilised on theair/liquid membrane contactor.

The free cells were cultivated exponentially at 0.2 h⁻¹ of specificgrowth rate up to the 18^(th) hour. The free biomass reached a maximumof 2.6 g l⁻¹ after one day of culture and then remained constant untilthe end of the culture.

Analysis of the gases revealed the presence of a biomass immobilised onthe membrane. The growth of this biomass took place during the first dayof culture in order to attain 1.2 g m⁻². A glucose consumption rate of1.61 g l⁻¹ h⁻¹ was measured during the first day of culture, and thenthis decreased as the glucose was depleted in the medium.

8.1.2 Analysis of the Mycosubtilin Produced

The production of mycosubtilin reached 10 mg l⁻¹ after two days ofculture. 45 mg was desorbed during washing of the membrane resulting ina total production of 25 mg l⁻¹ and a mean productivity of 0.5 mg l⁻¹h⁻¹. After purification by the microfiltration,ultrafiltration/diafiltration and drying steps, a mixture ofmycosubtilin in powder form containing the novel forms of mycosubtilinwas obtained. The purity of this mixture was more than 94%.

8.2 Continuous Production, Extraction and Purification of Mycosubtilinby B. subtilis BBG125 with Completely Recycled Cells

In this example, the B. subtilis BBG125 strain (filed on 10 Mar. 2011under the number CNCM I-4451 at the National Collection of MicroorganismCultures (CNCM) of the Institut Pasteur (Paris, France)), capable ofproducing only mycosubtilin in a constitutive manner, was cultivatedcontinuously at 22° C. for 72 hours in an air/liquid membrane contactorM1.

In addition to the air/liquid membrane contactor that allows the growthand immobilisation of the cells, the method presented in this example isalso characterised by the presence of:

-   -   devices for supplying and drawing off continuously in said        reactor a given flow of said substrate, in accordance with those        described in example 6,    -   a microfiltration membrane M2 with a surface area of 0.45 m² and        a pore size of 0.2 μm, in accordance with that described in        example 6,    -   three tanks: supply (tank 1), concentration (tank 2) and waste        (tank 3), in accordance with those described in example 6, and    -   an ultrafiltration membrane M3 with a surface area of 0.1 m² and        a cutoff threshold of 10 kDa, in accordance with that described        in example 6.

In addition, the supply and drawing-off rates of the pumps P1 and P5 inaccordance with those described in this example were equal and adjustedso as to obtain a degree of dilution of around 0.1 h⁻¹, that is to sayan hourly rate equal to 0.1 times the volume of the aqueous phasecontained in the production device.

One feature of this method in accordance with the present invention liesin the fact that all the microorganism cells in the production deviceare recycled after they have had the residues of organic matter andbiosurfactants removed, which makes it possible to obtain a high growthrate of the microorganisms on the membrane. The continuous culture waspreceded by 20 hours of batch culture.

8.2.1 Analysis of the Biomass

During the first 40 hours of the continuous culture at 0.1 h⁻¹ of degreeof dilution, the free biomass continued to increase in the broth up to7.2 g l⁻¹. Next, its concentration remained constant, which certainlyrevealed the presence of an inhibitor. On the other hand, analysis ofthe gases revealed a significant immobilisation of the cells on themembrane, which reached 3.1 g m⁻² after three days of culture. Duringthe first two days of culture, the concentration of glucose decreasedwith a glucose consumption rate of 1.5 g l⁻¹ h⁻¹.

8.2.2 Analysis of the Surfactin Produced

The mycosubtilin was thus extracted through the microfiltration membraneand was indeed concentrated by the 10 kDa ultrafiltration membrane; theaccumulation thereof in the intermediate tank was observed. Themycosubtilin productivity increased in the course of the first 40 hoursof culture and reached a maximum of 1.5 mg l⁻¹ h⁻¹. After thisexperiment, washing of the membranes recovered 84 mg of mycosubtilin. Atthe end of this experiment, the continuous culture produced 895 mg ofmycosubtilin in solution, that is to say an average concentrationproduced of 48 mg of mycosubtilin produced per litre of medium consumed.The ultrafiltration/diafiltration steps obtained surfactin in solutionwith a purity of 90%. This continuous method shows a productivity threetimes greater than that obtained with the batch mode described inexample 8.1.

LIST OF REFERENCES

-   [1] Ongena, M. and Jacques, P. 2008. Bacillus lipopeptides:    versatile weapons for plant disease biocontrol. Trends Microbiol.    16, 115-125.-   [2] CZ 20011620-   [3] DE 102005050123-   [4] FR 2578552-   [5] Guez, J. S. et al., 2007. Setting up and modelling of    overflowing fed-batch cultures of Bacillus subtilis for the    production and continuous removal of lipopeptides, J. Biotechnol.,    131, 67-75.-   [6] Davis, D. A., Lynch, H. C. and Varley, J., 1999. The production    of Surfactin in batch culture by Bacillus subtilis ATCC 21332 is    strongly influenced by the conditions of nitrogen metabolism. Enzyme    Microb. Technol. 25, 322-329.-   [7] WO 0226961-   [8] EP 1320595-   [9] Landy, M. et al. 1948. Bacillomycin; an antibiotic from Bacillus    subtilis active against pathogenic fungi. Proc. Soc. Exp. Biol. Med.    67, 539-541.-   [10] Guez, J. S. et al., 2008. Respiration activity monitoring    system (RAMOS), an efficient tool to study the influence of the    oxygen transfer rate on the synthesis of lipopeptide by Bacillus    subtilis. J. Biotechnol. 134, 121-126.-   [11] Remize, P. J. and Cabassud, C. 2003. A novel bubble-free    oxidation reactor: the G/L membrane contactor. Recent progress in    process engineering. Integration of membranes in the processes 2.    Lavoisier Tec et Doc.-   [12] Duitman, E. H. et al., 1999. The mycosubtilin synthetase of    Bacillus subtilis ATCC 6633: a multifunctional hybrid between a    peptide synthetase, an amino transferase, and a fatty acid synthase.    Proc. Natl. Acad. Sci. U.S.A., 96, 13294-13299.-   [13] Leclère, V. et al., 2005. Mycosubtilin overproduction by    Bacillus subtilis BBG100 enhances the organism's antagonistic and    biocontrol activities. Appl. Environ. Microbiol. 71, 4577-4584.-   [14] Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a    laboratory manual, 3^(rd) ed., Cold Spring Harbor Laboratory, Cold    Spring Harbor, N.Y.-   [15] Dennis, J. J. and Zylstra, G. J. 1998. Plasposons: modular    self-cloning minitransposon derivatives for rapid genetic analysis    of gram-negative bacterial genomes. Appl. Environ. Microbiol. 64,    2710-2715.-   [16] Herrero, M., de Lorenzo, V., and Timmis, K. N. 1990. Transposon    vectors containing non-antibiotic resistance selection markers for    cloning and stable chromosomal insertion of foreign genes in    gram-negative bacteria, J. Bacteriol. 172, 6557-6567.-   [17] Bertani, G. 2004. Lysogeny at mid-twentieth century: P1, P2,    and other experimental systems. J. Bacteriol. 186, 595-600.-   [18] Dubnau, D. A. 1982. Genetic transformation of bacillus subtilis    p 148-178. In D. A. Dubnau (Ed) The molecular biology of the    Bacilli, vol I. Bacillus subtilis. Academic Press, Inc. New York.-   [19] Besson, F. et al. 1979. Antifungal activity upon Saccharomyces    cerevisiae of iturin A, mycosubtilin, bacillomycin L and of their    derivatives; inhibition of this antifungal activity by lipid    antagonists. J. Antibiot. (Tokyo) 32, 828-833.-   [20] Chen, H. L., Chen, Y. S., and Juang, R. S. 2007. Separation of    surfactin from fermentation broths by acid precipitation and    two-stage dead-end ultrafiltration processes. J. Membr. Sci. 299,    114-121.-   [21] Chen, H. L., Chen, Y. S., and Juang, R. S. 2008. Flux decline    and membrane cleaning in cross-flow ultrafiltration of treated    fermentation broths for surfactin recovery. Sep. Purif. Technol. 62,    47-55.

1-15. (canceled)
 16. A population of genetically engineered Bacillus subtilis bacteria, wherein the promoter of the myc operon has been replaced by a constitutive promoter, and wherein the bacteria produce a mycosubtilin, the fatty acid chain of which comprises 17 carbon atoms and which has glutamine in place of asparagine in position 3 in its peptide cycle (C17 Gln3 mycosubtilin), represented by the following formula (II):


17. The population of genetically engineered Bacillus subtilis bacteria of claim 16, wherein the constitutive promoter is P_(repU).
 18. The population of genetically engineered Bacillus subtilis bacteria of claim 16, wherein the bacteria comprise a srfA operon that has been interrupted.
 19. The population of genetically engineered Bacillus subtilis bacteria of claim 18, which does not produce surfactin.
 20. A method of producing a population of genetically engineered Bacillus subtilis bacteria, the method comprising providing a parental strain of Bacillus subtilis bacteria, interrupting the srfA operon, and replacing the promoter of the myc operon with a constitutive promoter, such that the engineered bacteria does not produce any surfactin and produces a larger quantity of mycosubtilins as compared to the parental strain.
 21. The method of claim 20, wherein the parental strain of Bacillus subtilis bacteria is ATCC
 6633. 22. The method of claim 20, wherein the engineered bacteria produces C17 Gln3 mycosubtilin.
 23. A population of genetically engineered Bacillus subtilis bacteria produced by the method of claim
 20. 24. A Bacillus subtilis bacteria strain BBG125, which was deposited in the National Collection of Microorganism Cultures (CNCM) under number CNCM I-4451.
 25. A method of producing a mycosubtilin composition, the method comprising: providing the population of genetically engineered Bacillus subtilis bacteria of claim 16; culturing the bacteria in a culture medium comprising an organic substrate to produce a mycosubtilin composition; and recovering the mycosubtilin composition.
 26. The method of claim 25, wherein the mycosubtilin composition comprises 1%-20% C17 Gln3 mycosubtilin.
 27. The method of claim 26, further comprising assaying for the presence of 1%-20% C17 Gln3 mycosubtilin.
 28. The method of claim 25, wherein the organic substrate comprises starch, glucose, saccharose, glycerol, organic acids, amino acids, or a mixture thereof.
 29. The method of claim 25, wherein the organic substrate does not comprise xylose. 